Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus cajan (L.) Millspaugh]

BMC Plant Biology

Research Article

Tapas K Bandhopadhya¹  Douglas R Cook²  Ranjeet S Raje³  Subhojit Datta⁴  Mahendra N Singh⁵  Rajeev K Varshney⁶  Deepak K Gupta⁷  Sangeeta Singh⁷  Giriraj Kumawat⁷  Tilak R Sharma⁷  Vivek Dogra⁷  Nagendra K Singh⁷  Kishor Gaikwad⁷  Bikram P Singh⁷  Sutapa Dutta⁸  Pawan Kulwal⁹  KB Wanjari⁹  Fakrudin Bashasab^1,10 

[1] Department of Molecular Biology and Biotechnology, University of Kalyani, 741235, Kalyani, WB, India;Department of Plant Pathology, University of California, 95616-8680, Davis, CA, USA;Division of Genetics, Indian Agricultural Research Institute, 110012, New Delhi, India;Indian Institute of Pulses Research, 208024, Kanpur, UP, India;Institute of Agricultural Sciences, Banaras Hindu University, 221005, Varanasi, UP, India;International Crops Research Institute for the Semi-Arid Tropics, 502324, Patancheru, AP, India;National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, 110 012, New Delhi, India;National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, 110 012, New Delhi, India;Department of Molecular Biology and Biotechnology, University of Kalyani, 741235, Kalyani, WB, India;Panjabrao Deshmukh Krishi Vidyapeeth, Krishinagar, 444 104, Akola, Maharasthra, India;University of Agricultural Sciences, 580005, Dharwad, Karnataka, India;

关键词: Simple Sequence Repeat Marker;    Simple Sequence Repeat Locus;    Unigene Sequence;    Polymorphic Simple Sequence Repeat Marker;    Simple Sequence Repeat Polymorphism;   

DOI  :  10.1186/1471-2229-11-17

 received in 2010-04-29, accepted in 2011-01-20,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundPigeonpea [Cajanus cajan (L.) Millspaugh], one of the most important food legumes of semi-arid tropical and subtropical regions, has limited genomic resources, particularly expressed sequence based (genic) markers. We report a comprehensive set of validated genic simple sequence repeat (SSR) markers using deep transcriptome sequencing, and its application in genetic diversity analysis and mapping.ResultsIn this study, 43,324 transcriptome shotgun assembly unigene contigs were assembled from 1.696 million 454 GS-FLX sequence reads of separate pooled cDNA libraries prepared from leaf, root, stem and immature seed of two pigeonpea varieties, Asha and UPAS 120. A total of 3,771 genic-SSR loci, excluding homopolymeric and compound repeats, were identified; of which 2,877 PCR primer pairs were designed for marker development. Dinucleotide was the most common repeat motif with a frequency of 60.41%, followed by tri- (34.52%), hexa- (2.62%), tetra- (1.67%) and pentanucleotide (0.76%) repeat motifs. Primers were synthesized and tested for 772 of these loci with repeat lengths of ≥18 bp. Of these, 550 markers were validated for consistent amplification in eight diverse pigeonpea varieties; 71 were found to be polymorphic on agarose gel electrophoresis. Genetic diversity analysis was done on 22 pigeonpea varieties and eight wild species using 20 highly polymorphic genic-SSR markers. The number of alleles at these loci ranged from 4-10 and the polymorphism information content values ranged from 0.46 to 0.72. Neighbor-joining dendrogram showed distinct separation of the different groups of pigeonpea cultivars and wild species. Deep transcriptome sequencing of the two parental lines helped in silico identification of polymorphic genic-SSR loci to facilitate the rapid development of an intra-species reference genetic map, a subset of which was validated for expected allelic segregation in the reference mapping population.ConclusionWe developed 550 validated genic-SSR markers in pigeonpea using deep transcriptome sequencing. From these, 20 highly polymorphic markers were used to evaluate the genetic relationship among species of the genus Cajanus. A comprehensive set of genic-SSR markers was developed as an important genomic resource for diversity analysis and genetic mapping in pigeonpea.

【 授权许可】

Unknown   
© Dutta et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.